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The satellite RNA of bamboo mosaic virus (satBaMV) has a single open reading frame encoding a non-structural protein, P20, which facilitates long-distance movement of satBaMV in BaMV and satBaMV co-infected plants. Immunohistochemistry and immunoelectron microscopy revealed that the P20 protein accumulated in the cytoplasm and nuclei in co-infected cells. P20 and the helper virus coat protein (CP) were highly similar in their subcellular localization, except that aggregates of BaMV virions were not labelled with anti-P20 serum. The BaMV CP protein was fairly abundant in mesophyll cells, whilst P20 was more frequently detected in mesophyll cells and vascular tissues. The expression kinetics of the P20 protein was similar to but slightly earlier than that of CP in co-infectedBambusa oldhamiiprotoplasts andNicotiana benthamianaleaves. However, satBaMV-encoded protein levels declined rapidly in the late phase of co-infection. During co-infection, in addition to the intact P20, a low-molecular-mass polypeptide of 16 kDa was identified as a P20 C-terminally truncated product; the possible method of generation of the truncated protein is discussed.

The double-stranded telomeric repeat-binding protein (TRP) AtTRP1 is isolated from Arabidopsis thaliana. Using gel retardation assays, we defined the C-terminal 97 amino acid residues, Gln464 to Val560 (AtTRP1(464-560)), as the minimal structured telomeric repeat-binding domain. This region contains a typical Myb DNA-binding motif and a C-terminal extension of 40 amino acid residues. The monomeric AtTRP1(464-560) binds to a 13-mer DNA duplex containing a single repeat of an A.thaliana telomeric DNA sequence (GGTTTAG) in a 1:1 complex, with a K(D) approximately 10(-6)-10(-7) M. Nuclear magnetic resonance (NMR) examination revealed that the solution structure of AtTRP1(464-560) is a novel four-helix tetrahedron rather than the three-helix bundle structure found in typical Myb motifs and other TRPs. Binding of the 13-mer DNA duplex to AtTRP1(464-560) induced significant chemical shift perturbations of protein amide resonances, which suggests that helix 3 (H3) and the flexible loop connecting H3 and H4 are essential for telomeric DNA sequence recognition. Furthermore, similar to that in hTRF1, the N-terminal arm likely contributes to or stabilizes DNA binding. Sequence comparisons suggested that the four-helix structure and the involvement of the loop residues in DNA binding may be features unique to plant TRPs.

We have constructed a genetic linkage map for Nicotiana plumbaginifolia/Nicotiana longiflora (2n= 2x=20), based on the segregation of 69 RFLP and 102 RAPD loci in 99 F2 plants from the cross N. plumbaginifolia×N. longiflora. The map consists of nine major linkage groups, each containing more than nine marker loci, and spans 1062 cM. Twenty of the RFLP markers were mapped previously to Nicotiana sylvestris (2n=2x=24) chromosomes using monosomic alien addition lines. Taxonomically, N. plumbaginifolia and N. sylvestris belong to the same section, namely the Alatae; however, cytogenetic evidence indicates that they are not closely related. Comparison of the distribution of markers common to both maps suggests that genome reorganization has occurred during the evolution of these two species. Evidence is also presented that genome reorganization may be accompanied by gain and loss of specific classes of DNA sequences in their genomes.

A repetitive DNA sequence, ZmCR2.6c, was isolated from maize based on centromeric sequence CCS1 of the wild grass Brachypodium sylvaticum. ZmCR2.6c is 309 bp in length and shares 65% homology to bases 421–721 of the sorghum centromeric sequence pSau3A9. Fluorescence in situ hybridization (FISH) localized ZmCR2.6c to the primary constrictions of pachytene bivalents and to the stretched regions of MI/AI chromosomes, indicating that ZmCR2.6c is an important part of the centromere. Based on measurements of chromosome lengths and the positions of FISH signals of several cells, a pachytene karyotype was constructed for maize inbred line KYS. The karyotype agrees well with those derived from traditional analyses. Four classes of tandemly repeated sequences were mapped to the karyotype by FISH. Repeats 180 bp long are present in cytologically detectable knobs on 5L, 6S, 6L, 7L, and 9S, as well as at the termini and in the interstitial regions of many chromosomes not reported previously. A most interesting finding is the presence of 180-bp repeats in the NOR-secondary constriction. TR-1 elements co-exist with 180-bp repeats in the knob on 6S and form alone a small cluster in 4L. 26S and 5S rRNA genes are located in the NOR and at 2L.88, respectively. The combination of chromosome length, centromere position, and distribution of the tandem repeats allows all chromosomes to be identified unambiguously. The results presented form an important basis for using FISH for physical mapping and for investigating genome organization in maize.

Two tandemly repeated telomere-associated sequences, NP3R and NP4R, have been isolated from Nicotiana plumbaginifolia. The length of a repeating unit for NP3R and NP4R is 165 and 180 nucleotides respectively. The abundance of NP3R, NP4R and telomeric repeats is, respectively, 8.4 × 104, 6 × 103 and 1.5 × 106 copies per haploid genome of N. plumbaginifolia. Fluorescence in situ hybridization revealed that NP3R is located at the ends and/or in interstitial regions of all 10 chromosomes and NP4R on the terminal regions of three chromosomes in the haploid genome of N. plumbaginifolia. Sequence homology search revealed that not only are NP3R and NP4R homologous to HRS60 and GRS, respectively, two tandem repeats isolated from N. tabacum, but that NP3R and NP4R are also related to each other, suggesting that they originated from a common ancestral sequence. The role of these repeated sequences in chromosome healing is discussed based on the observation that two to three copies of a telomere-similar sequence were present in each repeating unit of NP3R and NP4R.

Large-size DNA of Nicotiana plumbaginifolia was manipulated in solution, concentrated by 2-butanol and cloned into a yeast artificial chromosome, pYACCMC5. A YAC library consisting of 22 000 clones with an average insert size of 190 kb has been constructed. The sizes of YAC inserts in this library ranged from 80–660 kb, indicating that 2-butanol can be used for concentrating large-size DNA with minimum shearing damage. This library represents 84% of the genome of N. plumbaginifolia . Characterization of this library revealed that only 6% of clones in the library contained sequences derived from chloroplast DNA. Some YAC inserts contained relatively long stretches of DNA, 100–180 kb, uninterrupted with repetitive sequences. Furthermore, both ends of most DNA molecules cloned in pYACCMC5 can be reisolated efficiently as recombinant plasmids in Escherichia coli . Approximately one third of the subcloned end fragments contained low- or single-copy DNA sequences. Single-copy end fragments are valuable probes to identify overlapping YAC clones by chromosome walking. The method for concentrating large-size DNA and the vector for YAC cloning described here should be useful in genome research programs. The YAC library presented here will facilitate mapping and cloning of genes from the genome of N. plumbaginifolia , which has been used for over a decade as a model species in plant cell genetics.

Rejuvenation, as evidenced by restored rooting competence, of Sequoia sempervirens by a series of repeated grafting of adult shoot tips onto juvenile rootstocks in vitro was confirmed. Complete restoration of rooting competence was achieved after grafting repeatedly five times. The rejuvenation was correlated with a disappearance of adult-associated esterase and peroxidase isozymes and an appearance of isoesterases and isoperoxidases that were characteristic of juvenile-phase shoots. These esterase and peroxidase isozymes could serve as markers to assist phasechange investigations.

Restriction fragment analysis revealed differences in mitochondrial DNA (mtDNA) between adult and juvenile coastal redwood, Sequoia sempervirens (D. Don) Endl. Thus, for example, juvenile shoots contained 4.0- and 3.6-kb BamHI], restricted fragments that were absent in adult material. Rejuvenation by 5 repetitive graftings onto juvenile rootstocks resulted in the same 4.0- and 3.6-kb fragments as were present in the juvenile material. The similarities in mtDNA fragments of juvenile and rejuvenated. shoots, as well as their distinction from adult shoots, were confirmed by double digestion, Southern hybridization and DNA sequencing.

The subtelomere and a portion of the associated telomeric region (together named 3RTAS) of chromosome IIIR from the Arabidopsis thaliana ecotypes Columbia (Col) and Wassilewskija (Ws) were specifically amplified by polymerase chain reaction and subsequently cloned and sequenced. The centromere-proximal portion of 3RTAS from both ecotypes contained two newly identified potential genes, one encoding the chloroplast luminal 19-kDa protein precursor and the other encoding three potential alternatively spliced CCCH-type zinc finger proteins. The telomere-proximal portion of 3RTAS from the Col ecotype contained short duplicated fragments derived from chromosomes I, II, and III, and that from the Ws ecotype contained a duplicated fragment derived from chromosome V. Each duplicated fragment has diverged somewhat in sequence from that of the ectopic template. Small patches of homologous nucleotides were found within the flanking sequences of both the duplicated fragments and the corresponding ectopic template sequences. The structural characteristics of these duplicated fragments suggest that they are filler DNAs captured by non-homologous end joining during double-strand break repair. Our characterization of 3RTAS not only filled up a gap in the chromosome IIIR sequence of A. thaliana but also identified new genes with unknown functions.

A gene (AtTRP1) encoding a telomeric repeat-binding protein has been isolated from Arabidopsis thaliana. AtTRP1 is a single copy gene located on chromosome 5 of A. thaliana. The protein AtTRP1 encoded by this gene is not only homologous to the Myb DNA-binding motifs of other telomere-binding proteins but also is similar to several initiator-binding proteins in plants. Gel retardation assay revealed that the 115 residues on the C terminus of this protein, including the Myb motif, are sufficient for binding to the double-stranded plant telomeric sequence. The isolated DNA-binding domain of AtTRP1 recognizes each telomeric repeat centered on the sequence GGTTTAG. The almost full-length protein of AtTRP1 does not form any complex at all with the DNA fragments carrying four or fewer GGTTTAG repeats. However, it forms a complex with the sequence (GGTTTAG)(8) more efficiently than with the sequence (GGTTTAG)(5). These data suggest that the minimum length of a telomeric DNA for AtTRP1 binding consists of five GGTTTAG repeats and that the optimal AtTRP1 binding may require eight or more GGTTTAG repeats. It also implies that this protein AtTRP1 may bind in vivo primarily to the ends of plant chromosomes, which consist of long stretches of telomeric repeats.

Forty-six callus cultures of rice (Oryza sativa L., 2n = 24), each presumably originating from a single microspore, were established and maintained on a medium containing 2,4-D. At the end of the first transfer 24% of the cultures were nonhaploid consisting of only diploid or polyploid cells, or of cells of two ploidy levels. Nuclear fusion and endomitosis occurring during the initial stages of in vitro microspore development were postulated to account for the formation of nonhaploid callus. Seventeen cultures were studied cytologically through 19 transfers. Only in one tetraploid and one hexaploid callus did the ploidy levels of cells remain unchanged during culture. Chromosome numbers in 13 cultures fell into a geometric series. Since no diplo- and quadruplochromosomes were observed, it was inferred that endomitosis rather than endoreduplication was responsible for the changes. In spite of the tendency for chromosome doubling, the proportions of cells of different ploidy levels were fixed in the 13 cultures at later transfers. Haploid cells were eliminated from all cultures. Diploid cells became predominant in eight cultures and tetraploid cells in five, suggesting a selection for either cell type. Triploids appeared in two cultures which initially did not contain this type of cell. Limited cytological information indicated that triploid cells might have originated from tetraploid cells through reductional grouping of chromosomes accompanied by multipolar formation.

The nucleotide sequence and the deduced primary structure of cellobiohydrolase II from Trichoderma reesei QM9414 have been determined. A palindromic region preceding this gene has a sequence common to the complementary strand of the putative eukaryotic cAMP dependent transcriptional signal sequence. The amino terminus of cellobiohydrolase II is homologous to the carboxy terminus of cellobiohydrolase I, endoglucanase I and endoglucanase II from the same organism. This region, which is rich in cysteine, glycine and serine, may represent the cellulose–binding domain of these proteins. The possible catalytic site of cellobiohydrolase II is also predicted.

Two families of tandem repeats, 180-bp and TR-1, have been found in the knobs of maize. In this study, we isolated 59 clones belonging to the TR-1 family from maize and teosinte. Southern hybridization and sequence analysis revealed that members of this family are composed of three basic sequences, A (67 bp); B (184 bp) or its variants B′ (184 bp), 2/3B (115 bp), 2/3B′ (115 bp); and C (108 bp), which are arranged in various combinations to produce repeat units that are multiples of ∼180 bp. The molecular structure of TR-1 elements suggests that: (1) the B component may evolve from the 180-bp knob repeat as a result of mutations during evolution; (2) B′ may originate from B through lateral amplification accompanied by base-pair changes; (3) C plus A may be a single sequence that is added to B and B′, probably via nonhomologous recombination; and (4) 69 bp at the 3′ end of B or B′, and the entire sequence of C can be removed from the elements by an unknown mechanism. Sequence comparisons showed partial homologies between TR-1 elements and two centromeric sequences (B repeats) of the supernumerary B chromosome. This result, together with the finding of other investigators that the B repeat is also fragmentarily homologous to the 180-bp repeat, suggests that the B repeat is derived from knob repeats in A chromosomes, which subsequently become structurally modified. Fluorescence in situ hybridization localized the B repeat to the B centromere and the 180-bp and TR-1 repeats to the proximal heterochromatin knob on the B chromosome.